Bottom Line:
PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear.The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition.These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs.

ABSTRACTType 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.

Figure 5: In vivo effects of PDE10A inhibition by TP-10. A, In the medial part of the dorsal striatum of drd2-EGFP adult mice treated with TP-10 (3 mg/kg), PH3 was selectively observed in D2 MSNs. In the lateral part of the dorsal striatum, PH3 immunoreactivity was observed in both EGFP-positive and EGFP-negative MSNs. EGFP and PH3 are shown in grayscale, and are overlaid with EGFP in green and PH3 in red (Merge). Scale bar, 20 µm. B, Each color spot represents a position where the relative distribution of D2/(D1 + D2) PH3-positive MSNs is indicated in pseudocolor, over a schematic of coronal mouse brain (Franklin and Paxinos, 2007). C, PH3-positive nuclei were quantified in medial and lateral parts of the dorsal striatum as defined by the dotted line in B. The effect of localization was significant (Kruskal–Wallis test followed by a Mann–Whitney test with a Dunn–Sidak adjustment test for pairwise multiple comparisons tests, p < 10−4), with PH3-positive nuclei being preferentially D2 MSNs in the medial striatum. ***indicates a difference between EGFP-positive (D2) and EGFP-negative (D1) MSNs with p < 10−4. D, The preferential AKAR3 response is also observed in the lateral striatum in brain slices from neonate mice. MSNs were transduced for the expression of the AKAR3 biosensor and imaged with wide-field microscope in the lateral striatum. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and was identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 µm) or CGS 21680 (CGS, 1 µm), respectively. The thick black line represents the average of all the traces in each group. TP-10 (100 nm) increased AKAR3 ratio selectively in D2 MSNs. E, The same experiment was repeated: there was no effect of localization, and TP-10 increased the AKAR3 ratio selectively in D2 MSNs in both the dorsolateral and dorsomedial striatum (two-way ANOVA: localization effect, F(1,12) = 0.374, p = 0.374; D1/D2 effect, F(1,12) = 44.01, p < 10−4; localization × D1/D2 interaction, F(1,12) = 0.042, p = 0.804. Bonferroni’s post hoc test: **p < 0.01.). C, E, Error bars indicate the SEM.

Mentions:
Two-photon imaging was used to separate individual neurons for a precise quantification of the amplitude of the response (Figs. 1, 2). Ratio measurements were performed on a series of 5–10 consecutive image from the image stack, centered on the cell body. With cytosolic biosensors, when visible, the nucleus was excluded from the measurement. Wide-field imaging (Figs. 3A–E) also allowed the unambiguous identification of D1 and D2 MSNs, provided that the infection level was kept low and no fluorescence overlap between neighboring neurons was observed. The optical cross-contamination resulting from out-of-focus light was evaluated by the final response to CGS 21680 and SKF-38393, applied sequentially: cells were rejected from analysis if the cross-contamination was >30%. For cGMP imaging (Fig. 3F,G), the data were quantified as relative ratio change.

Figure 5: In vivo effects of PDE10A inhibition by TP-10. A, In the medial part of the dorsal striatum of drd2-EGFP adult mice treated with TP-10 (3 mg/kg), PH3 was selectively observed in D2 MSNs. In the lateral part of the dorsal striatum, PH3 immunoreactivity was observed in both EGFP-positive and EGFP-negative MSNs. EGFP and PH3 are shown in grayscale, and are overlaid with EGFP in green and PH3 in red (Merge). Scale bar, 20 µm. B, Each color spot represents a position where the relative distribution of D2/(D1 + D2) PH3-positive MSNs is indicated in pseudocolor, over a schematic of coronal mouse brain (Franklin and Paxinos, 2007). C, PH3-positive nuclei were quantified in medial and lateral parts of the dorsal striatum as defined by the dotted line in B. The effect of localization was significant (Kruskal–Wallis test followed by a Mann–Whitney test with a Dunn–Sidak adjustment test for pairwise multiple comparisons tests, p < 10−4), with PH3-positive nuclei being preferentially D2 MSNs in the medial striatum. ***indicates a difference between EGFP-positive (D2) and EGFP-negative (D1) MSNs with p < 10−4. D, The preferential AKAR3 response is also observed in the lateral striatum in brain slices from neonate mice. MSNs were transduced for the expression of the AKAR3 biosensor and imaged with wide-field microscope in the lateral striatum. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and was identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 µm) or CGS 21680 (CGS, 1 µm), respectively. The thick black line represents the average of all the traces in each group. TP-10 (100 nm) increased AKAR3 ratio selectively in D2 MSNs. E, The same experiment was repeated: there was no effect of localization, and TP-10 increased the AKAR3 ratio selectively in D2 MSNs in both the dorsolateral and dorsomedial striatum (two-way ANOVA: localization effect, F(1,12) = 0.374, p = 0.374; D1/D2 effect, F(1,12) = 44.01, p < 10−4; localization × D1/D2 interaction, F(1,12) = 0.042, p = 0.804. Bonferroni’s post hoc test: **p < 0.01.). C, E, Error bars indicate the SEM.

Mentions:
Two-photon imaging was used to separate individual neurons for a precise quantification of the amplitude of the response (Figs. 1, 2). Ratio measurements were performed on a series of 5–10 consecutive image from the image stack, centered on the cell body. With cytosolic biosensors, when visible, the nucleus was excluded from the measurement. Wide-field imaging (Figs. 3A–E) also allowed the unambiguous identification of D1 and D2 MSNs, provided that the infection level was kept low and no fluorescence overlap between neighboring neurons was observed. The optical cross-contamination resulting from out-of-focus light was evaluated by the final response to CGS 21680 and SKF-38393, applied sequentially: cells were rejected from analysis if the cross-contamination was >30%. For cGMP imaging (Fig. 3F,G), the data were quantified as relative ratio change.

Bottom Line:
PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear.The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition.These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs.

ABSTRACTType 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.